Introduction

From a clinical standpoint, precise resection of hand tumors is of great importance since hands serve vital functions in everyday activities. Even small hand lesions can give rise to ongoing pain, noticeable deformities, or functional deficits, greatly diminishing patients’ quality of life. Among hand tumors, the most frequently encountered malignancy is squamous cell carcinoma (SCC), which accounts for 58%–90% of total cases (Mavrogenis et al., 2017; Valk et al., 2020). Given that surgical resection remains the mainstay of treatment for hand SCC, real-time intraoperative identification of SCC tumor margins is of critical significance. Overly aggressive resection may impair both the appearance and essential functions of the hand, whereas excessively conservative resection carries the risk of leaving residual tumor tissue. These retained tumor cells can subsequently invade adjacent structures, particularly regional lymph nodes. According to published data, approximately 14.3% of SCC patients develop distant metastases within 5 years, which is associated with worsened prognosis (Caud et al., 2023; Sayed et al., 2019). Current intraoperative assessment of resection margins mainly relies on frozen section analysis on selected tissue specimens, which involves a time-consuming multistep process (specimen collection, transportation, freezing, sectioning, staining, and pathological interpretation). This technique only provides information for a limited number of specimens, and its accuracy is highly dependent on the pathologist’s experience to distinguish cellular morphology. Furthermore, the formation of ice crystals during freezing may impair cellular integrity, thereby affecting accurate cell identification. Therefore, researchers worldwide have been exploring various alternative techniques for the real-time assessment of tumor margins.

Fluorescence-based approaches are proved to be excellent candidates for such applications owing to their high sensitivity and easy detectability (Refaat et al., 2022; Shcheslavskiy et al., 2025; Wang et al., 2024). Despite the abundance of fluorescent molecules, most traditional organic fluorophores always suffer from aggregation-caused quenching (ACQ) effect, which is characterized as emission diminishing or even complete quenching in aggregated states, severely restricting their utilizations in many cases. First proposed in 2001, the concept of aggregation-induced emission (AIE) has greatly reformed the conventional rules of fluorophore development. In contrast to ACQ fluorophores, aggregation-induced emission luminogens (AIEgens) exhibit weak or no emission in a dispersed state but become significantly luminescent upon molecular aggregation. This unique feature enables AIEgens as revolutionary materials for biosensing and bioimaging. Thus AIE was selected as the 2020 top ten emerging technologies in chemistry by IUPAC (Jiang et al., 2024; Luo et al., 2001; Mei et al., 2015). Although a wide variety of AIEgens have been reported to date, the development of systems with finely tunable emission wavelengths to meet diverse application demands remains highly significant (Kothavale and Sekar, 2017; Song et al., 2016; Xu et al., 2019). In this study, we conceived and developed a novel emission-tunable AIE platform based on a 1,4,5-triphenylimidazole core structure. This newly established platform, designated TPIT-X, features a donor–π–acceptor (D–π–A) backbone in which thiophene serves as an electron-rich π-linker. By simply introducing appropriate acceptor units, we synthesized a series of AIE emitters (TPIP-1 to TPIP-6) that exhibit broad emission windows ranging from green (490 nm) to deep red (695 nm), thereby covering almost the entire visible spectrum. Optical evaluations also confirmed their pronounced AIE characteristics. As a highly hopeful application method for AIE fluorophore, fluorescence-guided surgery (FGS) has been extensively studied in previous reports: following systemic administration of targeted contrast agents, the tumor region becomess highly luminescent. Surgeons can then either directly resect additional diseased tissue under fluorescence guidance or collect suspicious margin samples for further evaluation via paraffin section analysis. Inspired by these findings, we recognize that the direct application of fluorescence imaging in intraoperative pathological diagnosis is both promising and attractive, owing to its operational simplicity, rapid turnaround time and non-invasive nature, which largely circumvents the risk of adverse reactions. Importantly, in contrast to injectable fluorescent probes used in FGS, contrast agents employed for ex vivo detection are classified as Class III medical devices and do not require drug approval. Consequently, they can achieve faster market authorization with lower development risks. In consideration of the inherent superiority of AIE molecules with deep red emission for bioimaging, TPIT-6 was encapsulated with DSPE-PEG2000 and further conjugated with Anti epidermal growth factor receptor (Anti-EGFR) antibodies to prepare nanoparticles (NPs)-based fluorescent probes. Based on TPIT-6 NPs-Ab, we designed a fluorescence imaging-based method for rapid visualization of tumor margins, aiming to overcome the challenges that impede real-time pathological diagnosis during SCC surgery. In our workflow, freshly excised skin tissue samples were co-incubated with the EGFR-targeted TPIT-6 NPs-Ab fluorescent probe. This approach successfully enabled rapid localization of tumor boundaries within the tissue specimens under fluorescence imaging. In brief, our work not only establishes an innovative design strategy for AIEgens with wide color tunability but also provides a novel intraoperative pathological analysis technique for hand SCC tumor resection.